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Biomedical subjects

W W Cao

Publications and source records attributed to W W Cao.

At least 19 recordsLinked to original sources
PubMed2001

Molecular cloning, nucleotide sequence, and expression of genes encoding a polycyclic aromatic ring dioxygenase from Mycobacterium sp. strain PYR-1.

Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5' to the 3' direction, were a dehydrogenase, the dioxygenase small (beta)-subunit, and the dioxygenase large (alpha)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large alpha subunit did not cluster with most of the known alpha-subunit sequences but rather with three newly described alpha subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits.

Cloning, Molecular↗
PubMed2000

Cloning, sequencing, and expression in Escherichia coli of a cytochrome P450 gene from Cunninghamella elegans.

A polyclonal antibody against microsomes of a fungus, Cunninghamella elegans, was used to screen a C. elegans cDNA library. A cDNA clone, containing an open reading frame (ORF) encoding a protein of 389 amino acids (aa), was obtained. GenBank comparison (BLAST) showed that the protein was closely related to P450 because a heme-binding region, which is highly conserved in all P450 sequences, was found in the ORF protein. Using an oligo probe designed from this C. elegans heme-binding region to rescreen the cDNA library, we obtained three new clones. Sequence comparison showed that the three clones, with different length cDNA inserts, were from the same mRNA of the C. elegans P450 gene. One clone had the full C. elegans P450 gene, encoding 473 aa with a molecular mass of 54958.60, whereas the 389 was a part of the 473 aa without the N-terminal. The entire C. elegans P450 gene was successfully subcloned and overexpressed in a plasmid-Escherichia coli system (pQE30). Immunostaining with three antibodies (CYP1A1, CYP2E1, and CYP3A1) against mammalian P450 enzymes and benzidine staining for hemoproteins showed positive results for the recombinant protein expressed in E. coli. A phylogenetic tree was constructed by comparison of other fungal P450s to the C. elegans sequence. The C. elegans P450 clustered close to the cyp51 family and was named cyp509A1 by the International Committee on the Nomenclature for Cytochrome P450 Enzymes.

Amino Acid Sequence↗
PubMed2000

A rapid method for PCR detection of bovine materials in animal feedstuffs.

Rapid identification of bovine materials in animal feedstuffs is essential for effective control of a potential source of bovine spongiform encephalopathy. We have developed a rapid method for the detection of the presence of bovine materials in animal feeds. Animal feed samples were prepared by a Chelex-100 treatment method, then subjected to polymerase chain reaction (PCR) detection. The assay can be completed in 2 h including 30 min for sample preparation, 35-65 min for PCR cycling and 30 min for gel electrophoresis. This method is not only rapid, simple and consistent, but also avoids a hazardous waste disposal issue associated with a previously described guanidine thiocyanate (GuSCN) extraction-PCR method.

Animal Feed↗
PubMed2000

Cloning, expression, and characterization of the katG gene, encoding catalase-peroxidase, from the polycyclic aromatic hydrocarbon-degrading bacterium Mycobacterium sp. strain PYR-1.

A 81-kDa protein from Mycobacterium sp. strain PYR-1 was expressed in response to exposure of the strain to the polycyclic aromatic hydrocarbon pyrene and recovered by two-dimensional gel electrophoresis. The N-terminal sequence of the protein indicated that it was similar to catalase-peroxidase. An oligonucleotide probe designed from this sequence was used to screen a genomic library of Mycobacterium sp. strain PYR-1, and a positive clone, containing a part of the gene encoding the 81-kDa protein, was isolated. A gene-walking technique was used to sequence the entire gene, which was identified as katG for catalase-peroxidase. The deduced KatG protein sequence showed significant homology to KatGII of Mycobacterium fortuitum and clustered with catalase-peroxidase proteins from other Mycobacterium species in a phylogenetic tree. The katG gene was expressed in Escherichia coli to produce a protein with catalase-peroxidase activity. Since the induction of this catalase-peroxidase occurred in pyrene-induced cultures and the exposure of these cultures to hydrogen peroxide reduced pyrene metabolism, our data suggest that this enzyme plays a role in polycyclic aromatic hydrocarbon metabolism by strain PYR-1.

Bacterial Proteins↗
PubMed1999

Diagnosis of mouse hepatitis virus contamination in mouse population by using nude mice and RT-PCR.

Mouse hepatitis virus (MHV) infection in laboratory mouse populations is a serious problem, because the MHV infections are known to interfere with research results. Confirmation of indirect serological detection methods by viral isolation is difficult. Reverse transcription plus polymerase chain reaction (RT-PCR) was used to test 94 mouse tissue samples from suspected naturally MHV infected mice. Positive results were only obtained from two colon samples and one mixed sample with colon and liver. The low positive rate is probably due to the virus being rapidly cleared by the MHV antibodies produced in the mouse. However, RT-PCR detection of MHV in nude mice placed in the same cages with other non-nude mice or placed in cages with used dirty bedding, showed a very high positive rate: 10 out of 12 colon samples were positive (83%), and 5 out of 10 faecal samples were positive (50%). A single-tube, single step RT-PCR method and two procedures for isolation of the viral RNA for the RT-PCR assay were also included in this article.

Animals↗
PubMed1998

Identification and sequencing of a cDNA encoding 6-phosphogluconate dehydrogenase from a fungus, Cunninghamella elegans and expression of the gene in Escherichia coli.

The fungus, Cunninghamella elegans has been widely used in bioremediation and microbial models of mammalian studies in many laboratories. Using the polymerase chain reaction to randomly amplify the insert directly from the single non-blue plaques of a C. elegans cDNA library, then partly sequencing and comparing with GenBank sequences, we have identified a clone which contains C. elegans 6-phosphogluconate dehydrogenase gene. The polymerase chain reaction product was cloned into a plasmid, pGEM-T Easy vector for full insert DNA sequencing. The 6-phosphogluconate dehydrogenase gene (1458 bases) and the deduced protein sequence were determined from the insert DNA sequence. The gene was found by open reading frame analysis and confirmed by the alignment of the deduced protein sequence with other published 6-phosphogluconate dehydrogenase sequences. Several highly conserved regions were found for the 6-phosphogluconate dehydrogenase sequences. The 6-phosphogluconate dehydrogenase gene was subcloned and over-expressed in a plasmid-E. coli system (pQE30). The cell lysate of this clone has a very high 6-phosphogluconate dehydrogenase enzyme activity. Most of the recombinant protein in this system was formed as insoluble inclusion bodies, but soluble in high concentration of urea-buffer. Ni-NTA resin was used to purify the recombinant protein which showed 6-phosphogluconate dehydrogenase enzyme activity. The recombinant protein has a predicted molecular size correlating with that revealed by sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. The C. elegans 6-phosphogluconate dehydrogenase was in a cluster with yeast' 6-phosphogluconate dehydrogenase in the phylogenetic tree. Bacterial 6-phosphogluconate dehydrogenase and higher organisms' 6-phosphogluconate dehydrogenase were found in different clusters.

Amino Acid Sequence↗
PubMed1998

Quantification of cytokine mRNA in peripheral blood mononuclear cells using branched DNA (bDNA) technology.

Changes in the patterns of cytokine expression are thought to be of central importance in human infectious and inflammatory diseases. As such, there is a need for precise, reproducible assays for quantification of cytokine mRNA that are amenable to routine use in a clinical setting. In this report, we describe the design and performance of a branched DNA (bDNA) assay for the direct quantification of multiple cytokine mRNA levels in peripheral blood mononuclear cells (PBMCs). Oligonucleotide target probe sets were designed for several human cytokines, including TNFalpha, IL-2, IL-4, IL-6, IL-10, and IFNgamma. The bDNA assay yielded highly reproducible quantification of cytokine mRNAs, exhibited a broad linear dynamic range of over 3-log10, and showed a sensitivity sufficient to measure at least 3000 molecules. The potential clinical utility of the bDNA assay was explored by measuring cytokine mRNA levels in PBMCs from healthy and immunocompromised individuals. Cytokine expression levels in PBMCs from healthy blood donors were found to remain relatively stable over a one-month period of time. Elevated levels of IFNgamma mRNA were detected in PBMCs from HIV-1 seropositive individuals, but no differences in mean levels of TNFalpha or IL-6 mRNA were detected between seropositive and seronegative individuals. By providing a reproducible method for quantification of low abundance transcripts in clinical specimens, the bDNA assay may be useful for studies addressing the role of cytokine expression in disease.

Cytokines↗
PubMed1998

The effect of food restriction on the composition of intestinal microflora in rats.

The effect of a food-restricted diet on the fecal microflora of rats was studied by determining total anaerobic bacteria, bacterial cellular fatty acids, and the predominant intestinal bacteria shown by polymerase chain reaction (PCR) primers specific for the 16S rRNA gene sequences of 12 bacterial species. Twenty-four female Fischer 344 rats, 57 days old were divided into two groups and maintained on an NIH-31 diet. One group was fed ad libitum while the other group received 60% of ad libitum food intake (40% food restriction supplemented with vitamins and minerals equal to the ad libitum animals). After 2, 10, and 20 weeks on this dietary regimen, groups of four animals were sacrificed and the intestinal contents analyzed for changes in the bacterial flora. The anaerobic population for two-week (short-term) food-restricted rats was 3.2 x 10(8) per gram, slightly less than the 9.1 x 10(8) per gram found in the ad libitum-fed rats. The anaerobic populations in 20-week food restricted and ad libitum fed rats were 1.9 x 10(9) and 2.7 x 10(9) per gram, respectively. The total anaerobic population did not change significantly in either group during the 20-week study. No statistically significant differences were observed in the bacterial cellular fatty acid profiles between the two groups as determined by gas-liquid chromatography. PCR analysis of the intestinal contents indicated no significant shifts in the predominant flora due to dietary changes. The results, using three different methods to detect changes in the rat intestinal microflora, suggest that long-term dietary restriction had little effect on the microflora of female Fischer 344 rats.

Animals↗
PubMed1997

PCR detection of Ruminococcus spp. in human and animal faecal samples.

Polymerase chain reaction (PCR) procedures for Ruminococcus spp. were developed and used for the detection of Ruminococcus spp. in human and animal faeces. The PCR specificity was established for Ruminococcus albus, R. bromii, R. obeum, R. callidus and R. flavefaciens. The PCR sensitivity ranged from 4 to 100 cells of the pure cultures, depending on the species. Ruminococcus albus, R. bromii, R. obeum and R. callidus were detected in human faeces. R. bromii, R. albus and R. obeum were also detected in animal faeces. R. flavefaciens was not detected in most of the samples tested, except for a weak positive in monkey faecal specimens. The PCR appears to be a specific and efficient method for the detection of Ruminococcus spp. in faecal specimens.

Animals↗
PubMed1997

Phylogenetic analysis and identification of Shigella spp. by molecular probes.

Oligonucleotide probes were used for identification of Shigella and analysis of the relationship between Shigella spp. and Escherichia coli. Probe-based PCRs shown cross-reactions from Shigella to E. coli. Probe-based 16S rRNA sequencing and phylogenetic analysis showed the four species of Shigella: Sh. dysenteriae, Sh. boydii, Sh. sonnei, and Sh. flexneri, formed a cluster with E. coli. Shigella flexneri and Sh. sonnei are even more similar to E. coli than to the other two Shigella species. These results confirmed an earlier recommendation that the four species of Shigella and E. coli should be classified as five sub-groups within a single species.

DNA Probes↗
PubMed1997

A universal protocol for PCR detection of 13 species of foodborne pathogens in foods.

A universal protocol for PCR detection of 13 species of foodborne pathogens in foods was developed. The protocol used a universal culture medium and the same PCR conditions with 13 sets of specific primers. The 13 species of foodborne pathogens examined were Escherichia coli, E. coli-ETEC, E. coli-O157:H7, Shigella spp., Salmonella spp., Yersinia enterocolitica, Y. pseudotuberculosis, Vibrio cholerae, V. parahaemolyticus, V. vulnificus, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus. No interference was observed using the PCR assay when food sample was artificially inoculated with each individual bacterial species. Twelve different seafood samples and two soft cheese samples without artificial inoculation were examined by this protocol. Vibrio vulnificus, Salmonella spp., E. coli, Listeria monocytogenes and Bacillus cereus were detected in some foods. Internal probe hybridization and nested PCR procedures were used to confirm the above findings.

Bacteria↗
PubMed1996

Phylogenetic analysis of Fusobacterium prausnitzii based upon the 16S rRNA gene sequence and PCR confirmation.

In order to develop a PCR method to detect Fusobacterium prausnitzii in human feces and to clarify the phylogenetic position of this species, its 16S rRNA gene sequence was determined. The sequence described in this paper is different from the 16S rRNA gene sequence is specific for F. prausnitzii, and the results of this assay confirmed that F. prausnitzii is the most common species in human feces. However, a PCR assay based on the original GenBank sequence was negative when it was performed with two strains of F. prausnitzii obtained from the American Type Culture Collection. A phylogenetic tree based on the new 16S rRNA gene sequence was constructed. On this tree F. prausnitzii was not a member of the Fusobacterium group but was closer to some Eubacterium spp. and located between Clostridium "clusters III and IV" (M.D. Collins, P.A. Lawson, A. Willems, J.J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J.A.E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994).

Base Sequence↗
PubMed1996

Reclassification of a polycyclic aromatic hydrocarbon-metabolizing bacterium, Beijerinckia sp. strain B1, as Sphingomonas yanoikuyae by fatty acid analysis, protein pattern analysis, DNA-DNA hybridization, and 16S ribosomal DNA sequencing.

A bacterium isolated from a polluted stream, capable of metabolizing biphenyl, naphthalene, phenanthrene, and higher-molecular-weight polycyclic aromatic hydrocarbons (D. Gibson, V. Mahadevan, D. Jerina, H. Yagi, and H. Yeh, Science 189:295-297, 1975), was previously identified as Beijerinckia sp. strain B1. In this investigation, 16S rRNA gene sequencing, biochemical tests, fatty acid methyl ester analysis, polyacrylamide gel electrophoresis of protein, and DNA-DNA hybridization were used to determine the taxonomic relationship of Beijerinckia sp. strain B1. The sequence of the 16S rRNA gene of B1 was identical to that of Sphingomonas yanoikuyae ATCC 51230T. The biochemical tests, fatty acid analysis, and sodium dodecyl sulfate-polacrylamide gel electrophoresis profile of soluble proteins of strain B1 showed results similar to those of S. yanoikuyae. DNA-DNA hybridization indicated that B1 and S. yanoikuyae ATCC 51230T are 75% homologous at the DNA level. We propose that Beijerinckia sp. strain B1 be reclassified as S. yanoikuyae.

Bacterial Proteins↗
PubMed1996

PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples.

PCR procedures based on 16S rRNA gene sequences specific for 12 anaerobic bacteria that predominate in the human intestinal tract were developed and used for quantitative detection of these species in human (adult and baby) feces and animal (rat, mouse, cat, dog, monkey, and rabbit) feces. Fusobacterium prausnitzii, Peptostreptococcus productus, and Clostridium clostridiiforme had high PCR titers (the maximum dilutions for positive PCR results ranged from 10(-3) to 10(-8)) in all of the human and animal fecal samples tested. Bacteroides thetaiotaomicron, Bacteroides vulgatus, and Eubacterium limosum also showed higher PCR titers (10(-2) to 10(-6)) in adult human feces. The other bacteria tested, including Escherichia coli, Bifidobacterium adolescentis, Bifidobacterium longum, Lactobacillus acidophilus, Eubacterium biforme, and Bacteroides distasonis, were either at low PCR titers (less than 10(-2)) or not detected by PCR. The reported PCR procedure including the fecal sample preparation method is simplified and rapid and eliminates the DNA isolation steps.

Adult↗
PubMed1996

Purification of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83 and characterization of the gene (bphC).

The 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) of Pseudomonas putida OU83 was constitutively expressed and purified to apparent homogeneity. The apparent molecular mass of the native enzyme was 256 kDa, and the subunit molecular mass was 32 kDa. The data suggested that 2,3-DBPD was an octamer of identical subunits. The nucleotide sequence of a DNA fragment containing the bphC region was determined. The deduced protein sequence for 2,3-DBPD consisted of 292 amino acid residues, with a calculated molecular mass of 31.9 kDa, which was in agreement with data for the purified 2,3-DBPD. Nucleotide and amino acid sequence analyses of the bphC gene and its product, respectively, revealed that there was a high degree of homology between the OU83 bphC gene and the bphC genes of Pseudomonas cepacia LB400 and Pseudomonas pseudoalcaligenes KF707.

Amino Acid Sequence↗
PubMed1996

Detection of Pasteurella pneumotropica in laboratory mice and rats by polymerase chain reaction.

A 16S rDNA-based polymerase chain reaction (PCR) method specific for Pasteurella pneumotropica was developed. The PCR product, a 395-base pair DNA fragment, was amplified from P. pneumotropica and not from 42 other bacterial species tested, including four other Pasteurella species and Actinobacillus ureae. The PCR method was used to identify 13 previously isolated strains that had been identified as P. pneumotropica by conventional methods: 12 were confirmed by PCR; one that was PCR-negative was re-examined by biochemical methods and determined to be A. ureae. The PCR detection of P. pneumotropica in nasopharyngeal swab specimens from 121 surveillance animals (15 inbred mice and 5 inbred rats from 20 animal rooms) had a high carrier state in healthy laboratory animals; for example, rat swab specimens were 89.6% (43/48) positive by PCR, 8.3% were positive by the direct culture-biochemical method, and 16.7% were positive by the enrichment culture-biochemical method. The positive rate for mice (21.9% [16/73]) was lower than that for rats.

Animals↗
PubMed1995

Phylogenetic analysis of polycyclic aromatic hydrocarbon degrading mycobacteria by 16S rRNA sequencing.

Mycobacterium sp. PYR-1 was previously isolated in our laboratory and was shown to be able to mineralize high molecular mass polycyclic aromatic hydrocarbons (PAHs) [Heitkamp and Cerniglia, (1988) Appl. Environ. Microbiol. 54, 1612-1614]. In this research, the 16S rRNA gene (rDNA) of this strain was amplified by polymerase chain reaction (PCR) and directly sequenced by cycle sequencing method. We compared this sequence with all known mycobacterial 16S rDNA sequences available from GenBank and found that Mycobacterium sp. PYR-1 16S rDNA differs from the other mycobacteria, especially in the region of nucleotides 168-200 (in the Escherichia coli numbering system). Using the 16S rDNA sequences of the mycobacteria, a phylogenetic tree was constructed. The data from the phylogenetic tree and similarity values suggest that Mycobacterium sp. PYR-1 is closer to M. aurum and M. vaccae. Using the same approach, we also determined the 16S rDNA from an another PAH-degrading Mycobacterium sp. PAH135, isolated by Grosser and colleagues (1991) (Appl. Environ. Microbiol. 57, 3462-3469). Mycobacterium sp. PAH135 was found to be closer to M. aichiense, and different from our Mycobacterium sp. PYR-1.

Base Sequence↗